The present invention relates to a composition and method to improve the storage of platelets prior to transfusion into a patient. Platelets are obtained as a by-product from whole blood donations and from plateletpheresis procedures. Typically, they are stored at 22.degree..+-.2.degree. C. in their own plasma within a plastic container whose walls are permeable to atmospheric gases. The plasma associated with these platelets normally contains all the=ingredients of normal plasma plus ingredients in the primary anticoagulant which result in a dextrose concentration five times the physiologic concentration. The dextrose is added to the primary anticoagulant for the benefit of red cells which require it during storage, and dextrose is generally accepted to be required for platelet storage as well. In routine blood banking practice, thee primary anticoagulant which is utilized is citrate-phosphate-dextrose (CPD), from Mollison, P. L., Blood Transfusion in Clinical Medicine, 7th Edition, Blackwell, 1983.
Donations of a unit of blood (63 ml of CPD mixed with 450 ml of whole blood) are processed by centrifugation into three fractions: red cells, plasma, and platelets. The volume of packed red cells from a unit of blood is approximately 180 ml, with a remaining volume of plasma and anticoagulant of about 333 ml. As used in the remainder of this application, the term plasma includes any anticoagulant which has been added thereto at the time of blood collection. The red cells are typically suspended in approximately 45 ml of plasma. Platelets are suspended in approximately 50 ml of plasma. This platelet containing product is typically referred to as a "platelet concentrate" (PC). The remaining 238 ml of plasma is frozen as fresh plasma. New techniques for preparing platelets for transfusion include platelet pheresis and the "buffy coat technique". During platelet pheresis, a single donor provides about five units of platelets by allowing the blood to be withdrawn and processed by a pheresis machine which separates the platelets for storage and redirects plasma, and optionally also the red cells, back to the donor. The "buffy coat technique" allows for the pooling of the platelets from several donors, usually about 4-6 donors, with the subsequent storage of the platelets in a storage medium that is either a plasma-based medium or a synthetic-based medium.
A great deal is known about human platelet cells. General papers describing techniques, materials and methods for storage of platelets are described by Murphy et al. in "Improved Storage of Platelets for Transfusion in a New Container", Blood, 60(1):194-200 (July, 1982); by Murphy in "The Preparation and Storage of Platelets for Transfusion", Mammon, Barnhart, Lusher and Walsh, PJD Publications, Ltd., Westbury, New York (]980);by Murphy in "Platelet Transfusion", Progress in Hemostasis and Thrombosis, Vol. III, Edited by Theodore Spaet, Grune and Stratton, Inc. (1976); by Murphy et al. in "Platelet Storage at 22.degree. C.: Role of Gas Transport Across Plastic Containers in Maintenance of viability", Blood, 46(2) 209-218 (1975); by Kilkson, Holme and Murphy in "Platelet Metabolism During Storage of Platelet Concentrates at 22.degree. C.", Blood, 64 (2) 406-414 (1984); by Murphy in "Platelet Storage for Transfusion", Seminars in Hematology, 22(3) 165-177 (1985); by Simon, Nelson, Carmen,and Murphy in "Extension of Platelet Concentrate Storage", Transfusion, 23:207-212 (1983); by Cesar, Diminno, Alam, Silver and Murphy in "Plasma Tree Fatty Acid Metabolism During Storage of Platelet Concentrates for Transfusion", Transfusion, 27(5) :434-437 (1987); each of which publications is hereby incorporated by reference and is more fully set forth herein.
There exists a considerable body of prior art concerning storage of platelets. Prior work has shown that the duration of platelet storage is limited by the continuing production of lactic acid from dextrose by the platelets. Although this provides energy for the platelets, the lactic acid acidifies the medium, which acidity eventually destroys the platelets. It has also been shown that platelets consume oxygen during storage for energy production, the end product of which process is a gas, CO.sub.2, which unlike lactic acid, can leave the platelet concentrate through the plastic walls of the container in which it is normally stored. The production of CO.sub.2 does not acidify the storage medium for the platelets. In addition to the glycolysis of dextrose, fatty acids and amino acids typically present in the plasma may be used as substrates for oxidative metabolism of stored platelet cells.
Most platelet storage media contain glucose. In U.S. Pat. No. 4,695,460 (Holme), a synthetic platelet storage media is disclosed containing 3.0-7.5 grams of dextrose, 3.0-6.0 grams of sodium citrate, and 2.0-4.2 grams of sodium bicarbonate. U.S. Pat. No. 4,447,415 (Rock) discloses a number of platelet storage solutions. It has been appreciated that platelet storage in a medium essentially free of glucose could be advantageous. For example, in U.S. Pat. No. 4,828,976, Murphy discloses a glucose free media for storing blood platelets. To store platelets for periods in excess of 5 days, it is taught that the storage media should be essentially free of glucose. It is also disclosed that it is the presence of glucose that leads to the generation of lactic acid which adversely affects platelet viability.
The rapid loss of platelet function during storage presents a significant problem in blood banking. One approach for diminishing or delaying the loss of platelet function during storage has been the development of plasma-free storage media. For example, U.S. Pat. No. 4,695,460 (Holme) and U.S. Pat. No. 4,447,414 (Rock et al.).
Another approach has focused on the biochemistry of platelet activation and means to regulate platelet activation, which results in platelet lysis and death. U.S. Pat. No. 4,994,367 (Bode et al.) discloses a blood platelet preparation comprising blood platelets, an adenylate cyclase stimulator (Prostaglandin El), a phosphodiesterase inhibitor (Theophylline), a thrombin inhibitor(N-(2-naphthylsulfonylglycyl)-D,L-amidinophenylalaninpiperidide), and a plasmin inhibitor (Aprotinin). A plasma-free platelet storage medium comprising dextrose, sodium citrate, sodium bicarbonate, and a platelet activation inhibitor (adenylate cyclase stimulator) is also disclosed. A process for preparing a plasma-free platelet preparation by producing platelet-rich plasma (PRP) from whole blood, adding a platelet activation inhibitor (adenylate cyclase stimulator), centrifuging the PRP to deposit the platelets on the bottom of the centrifuge container, removing the platelet-free plasma supernatant, and adding a plasma-free liquid platelet storage medium is also provided.
The adenylate cyclase stimulator is included to increase the production of adenosine 3',5'-cyclic phosphate (cAMP) in the blood platelets. The phosphodiesterase inhibitor is included to reduce the degradation of cAMP in the blood platelets. The thrombin inhibitor is included to reduce the stimulation of the blood platelets. The plasmin inhibitor is included to reduce the degradation of cell surface proteins on the blood platelets.
Heaton et al., British Journal of Hematology, 75:400-407 (1990) disclose an ionically balanced electrolyte solution with citrate, glucose and bicarbonate which was shown to provide good platelet viability with storage for up to 7 days When adenine was added to this solution, it also allowed for satisfactory preservation of red cells for extended periods.
A major problem of platelet storage in synthetic media is the potential for phi fall resulting from the lactate end product of glycolytic energy metabolism. The approach taken here is to add bicarbonate to buffer the acid load generated by a glucose-containing medium. In vivo studies demonstrated improved post-transfusion viability with platelets stored in this medium as compared to CPD-plasma.
Guppy et al., Vox Sanguinis, 59:146-152 (1990) studied the metabolism of platelets in vitro. It was found that glucose is never oxidized to any significant extent and is always converted to lactate, regardless of oxygen availability. Preliminary storage experiments using plasma-free media showed that an acetate-containing buffered salt solution provided excellent storage conditions and that a medium without any exogenous fuel is better than one containing glucose. Thus, it is concluded that a platelet storage medium should contain minimal amounts of glucose, and an oxidizable fuel in order to supplement the endogenous one. The identity of this fuel is not known; however, it is shown not to be glucose or glycogen. It is concluded that platelets can use acetate and when present, this fuel completely replaces the endogenous fuel. The data suggest that acetate, short chain fatty acids or amino acids metabolizable through the TCA cycle should provide ATP efficiently at low molarities without producing toxic end-products.
Notwithstanding the considerable work conducted in this area, a need still exists for means to improve the storage of platelets in a viable condition.